Hepatic lipase, a heparin-binding glycoprotein, functions in the metabolism of circulating lipoproteins. Hepatic lipase has been implicated in both processing of high-density lipoproteins, thus playing a role in atherogenesis, and metabolism of very low density lipoproteins and chylomicrons. The enzyme level is affected by a number of hormones as well as alcohol and vitamin A. Secondary hepatic lipase deficiency is seen accompanying a number of different diseases. The cDNA for rat hepatic lipase has been recently cloned. The protein has a molecular weight of 53K and contains two N- glycosylation sites. The sequence contains regions of homology with other known lipases, including two regions which are homologous to a sequence which has been shown to participate in lipid binding. These lipid-binding regions will be separately mutated using cassette mutagenesis. Normal and mutant proteins will be produced using an expression vector. The proteins will be assayed for activity using various substrates. The results from these experiments will resolve whether the binding regions have different affinities for various lipids and lipoproteins, and allow conclusions concerning the mode of enzyme action and the nature of the physiological substrate. The hepatic lipase heparin binding site will be characterized. Different regions of the cDNA clone will be subcloned into a vector to produce fusion proteins. The proteins will be tested for their ability to bind to heparin-Sepharose. Further subcloning of the binding region will completely define the site. The site will be tested for its ability to bind isolated liver endothelial cells. The effects of steroid hormones on hepatic lipase levels will be determined. Hep-G2 cells and rats will be treated with steroid hormones, and enzyme activity and mRNA levels will be measured. It will be possible to determine if the hormone effects are at the level of transcription. If this is the case, the gene for hepatic lipase will be isolated and control of gene expression will be further investigated. The regulatory roles of free fatty acid and various lipoproteins will be investigated in similar experiments.